Discovery of deeply intronic damaging mutations with nanopore sequencing

Abstract

Deeply intronic pathogenic variants are under-reported for several reasons: they are not reported by gene panels; they may lie within repetitive regions not aligned by short reads; in silico prediction tools have low precision for these variants; and most RNA testing is limited to evaluation of variants within ~20 bp of exon-intron junctions. We have developed a multiplexed targeted long-read DNA and cDNA sequencing approach, based on patient-derived DNA and RNA, that systematically evaluates deep intronic variants. We simultaneously identify all SNVs and small indels across the full genomic loci of targeted genes by adaptive sampling; apply in silico tools to predict variants that may create donor or acceptor splice sites; then use gene-specific cDNA sequencing to identify abnormal transcripts. In BRCA1 and ATM genes, we have identified pathogenic variants that create deeply intronic pseudo-exons that are spliced into transcripts, introducing premature truncations leading to loss of gene function.

Biography

Tom Walsh earned his PhD from the University of Leicester; following this he was a post-doctoral researcher at the University of Washington in Seattle, with Mary-Claire King, PhD. He is currently Research Professor of Medical Genetics at the University of Washington, working on inherited breast and ovarian cancer, using new and emerging genomic and sequencing technologies.